Activated coagulation factor IX (fIXa) is a key component of the coagulation cascade involved in fX activation. Deficiency of fIXa is central to the pathogenesis of hemophila B, while fIXa activity is implicated in coagulopathies associated with venous thrombosis, stroke, and atherosclerosis. The molecular mechanisms underlying the regulation of fIXa are poorly understood. The proposed studies will address this critical gap in our understanding of the physiology and pathophysiology of coagulation. We hypothesize that synergy between effects of cofactor and substrate and binding to exosites on fIXa results in productive alteration of the fIXa active site. Our long-term goal is to elucidate these effects and those of modulators implicated in pathophysiologic coagulation and use this information to define the perturbations that occur in fIXa upon formation of the fully assembled fX activation complex. We will use cofactor, fX, fX domain, and heparin to dissect effects of exosite occupation on fIXa. 3 independent approaches will be used to address the following 3 Specific Aims: 1) To define the structural requirements regulating the procoagulant function of fIXa using Kunitz-type inhibitors to modify its active site. 2) To define cofactor-dependent perturbations in the fIXa active site and activity, and 3) To define substrate-dependent perturbations in the fIXa active site activity that regulate fIXa activity through substrate-assisted catalysis. While the Kunitz-type inhibitor protease nexin-2 (PN-2 KPI) is a potent inhibitor of fIXa, the related bovine pancreatic trypsin inhibitor (BPTI) is not. In Aim 1, we will use a structure-based approach and interchange regions of BPTI and PN-2 KPI to define active site determinants in fIXa. Additionally, we have found that heparin promotes the reactivity of fIXa with both a pentapeptide substrate and BPTI, but not PN-2 KPI. In Aims 2 &3, we will merge combinatorial peptide chemistry and phage display approaches to perform comparative analyses of preferred substrate and inhibitor sequences for fIXa alone versus fIXa in complex with cofactor, heparin, and substrate mimics. These results will be confirmed via mutagenesis of fIXa and rescreening libraries to determine regions of fIXa responsive to modulation. These studies will define a novel paradigm by which cofactor and substrates synergistically modify the active site and procoagulant function. As such, this work will foster the development of novel therapeutics for either coagulopathies or bleeding disorders.